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Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

Christensen LA, Wang H, Van Houten B, Vasquez KM - Nucleic Acids Res. (2008)

Bottom Line: Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease.In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone.Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB.

View Article: PubMed Central - PubMed

Affiliation: Department of Carcinogenesis, Science Park-Research Division, University of Texas MD Anderson Cancer Center, Smithville, TX, USA.

ABSTRACT
Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

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The target duplex and psoralen-modified TFO. (A) Space-filling model of a psoralen-modified TFO bound to its target duplex. The psoralen moiety is shown in yellow and the TFO is shown in red bound to the purine-rich strand of the target duplex (blue) in the major groove. The pyrimidine-rich strand is shown in green (21). (B) Psoralen-modified TFO and TFO binding site. The TFO binding site is shown in red, the psoralen crosslinking site is shown in yellow and the pyrimidine-rich (py) and purine-rich (pu) strands are depicted in green and blue, respectively. The sequence of the TFO binding site is shown in bold capital letters, the psoralen crosslinking site is underlined and psoAG30, the psoralen-conjugated TFO, is depicted in an antiparallel orientation relative to the purine-rich strand of the target duplex. The MluI restriction site was used to remove the 3′-radiolabel on the pyrimidine strand. Figure 1A reprinted with permission from Vasquez et al., Biochemistry, 35, 10712–10719, Copyright 1996 American Chemical Society.
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Figure 1: The target duplex and psoralen-modified TFO. (A) Space-filling model of a psoralen-modified TFO bound to its target duplex. The psoralen moiety is shown in yellow and the TFO is shown in red bound to the purine-rich strand of the target duplex (blue) in the major groove. The pyrimidine-rich strand is shown in green (21). (B) Psoralen-modified TFO and TFO binding site. The TFO binding site is shown in red, the psoralen crosslinking site is shown in yellow and the pyrimidine-rich (py) and purine-rich (pu) strands are depicted in green and blue, respectively. The sequence of the TFO binding site is shown in bold capital letters, the psoralen crosslinking site is underlined and psoAG30, the psoralen-conjugated TFO, is depicted in an antiparallel orientation relative to the purine-rich strand of the target duplex. The MluI restriction site was used to remove the 3′-radiolabel on the pyrimidine strand. Figure 1A reprinted with permission from Vasquez et al., Biochemistry, 35, 10712–10719, Copyright 1996 American Chemical Society.

Mentions: The 120-bp duplex target was designed to contain a 30-bp TFO binding site and a psoralen 5′-AT-3′ crosslinking site (Figure 1). TFO-ICL substrate was created by incubating the 120-bp duplex with the psoralen-modified TFO (psoAG30) followed by irradiation with UVA at 1.8 J/cm2 to crosslink the psoralen to the target duplex. ICL-only substrate was constructed using a psoralen-disulfide-linked TFO (pso-SS-AG30). Following incubation with pso-SS-AG30 and UVA irradiation, the sample was treated with DTT to reduce the disulfide bond to release the TFO. Crosslinked samples were then purified by denaturing PAGE. The fluorescein-adducted duplex F2650, which has previously been shown to be efficiently incised by UvrABC nuclease (15,16), was used as a positive control.Figure 1.


Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

Christensen LA, Wang H, Van Houten B, Vasquez KM - Nucleic Acids Res. (2008)

The target duplex and psoralen-modified TFO. (A) Space-filling model of a psoralen-modified TFO bound to its target duplex. The psoralen moiety is shown in yellow and the TFO is shown in red bound to the purine-rich strand of the target duplex (blue) in the major groove. The pyrimidine-rich strand is shown in green (21). (B) Psoralen-modified TFO and TFO binding site. The TFO binding site is shown in red, the psoralen crosslinking site is shown in yellow and the pyrimidine-rich (py) and purine-rich (pu) strands are depicted in green and blue, respectively. The sequence of the TFO binding site is shown in bold capital letters, the psoralen crosslinking site is underlined and psoAG30, the psoralen-conjugated TFO, is depicted in an antiparallel orientation relative to the purine-rich strand of the target duplex. The MluI restriction site was used to remove the 3′-radiolabel on the pyrimidine strand. Figure 1A reprinted with permission from Vasquez et al., Biochemistry, 35, 10712–10719, Copyright 1996 American Chemical Society.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602775&req=5

Figure 1: The target duplex and psoralen-modified TFO. (A) Space-filling model of a psoralen-modified TFO bound to its target duplex. The psoralen moiety is shown in yellow and the TFO is shown in red bound to the purine-rich strand of the target duplex (blue) in the major groove. The pyrimidine-rich strand is shown in green (21). (B) Psoralen-modified TFO and TFO binding site. The TFO binding site is shown in red, the psoralen crosslinking site is shown in yellow and the pyrimidine-rich (py) and purine-rich (pu) strands are depicted in green and blue, respectively. The sequence of the TFO binding site is shown in bold capital letters, the psoralen crosslinking site is underlined and psoAG30, the psoralen-conjugated TFO, is depicted in an antiparallel orientation relative to the purine-rich strand of the target duplex. The MluI restriction site was used to remove the 3′-radiolabel on the pyrimidine strand. Figure 1A reprinted with permission from Vasquez et al., Biochemistry, 35, 10712–10719, Copyright 1996 American Chemical Society.
Mentions: The 120-bp duplex target was designed to contain a 30-bp TFO binding site and a psoralen 5′-AT-3′ crosslinking site (Figure 1). TFO-ICL substrate was created by incubating the 120-bp duplex with the psoralen-modified TFO (psoAG30) followed by irradiation with UVA at 1.8 J/cm2 to crosslink the psoralen to the target duplex. ICL-only substrate was constructed using a psoralen-disulfide-linked TFO (pso-SS-AG30). Following incubation with pso-SS-AG30 and UVA irradiation, the sample was treated with DTT to reduce the disulfide bond to release the TFO. Crosslinked samples were then purified by denaturing PAGE. The fluorescein-adducted duplex F2650, which has previously been shown to be efficiently incised by UvrABC nuclease (15,16), was used as a positive control.Figure 1.

Bottom Line: Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease.In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone.Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB.

View Article: PubMed Central - PubMed

Affiliation: Department of Carcinogenesis, Science Park-Research Division, University of Texas MD Anderson Cancer Center, Smithville, TX, USA.

ABSTRACT
Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

Show MeSH
Related in: MedlinePlus